NR1H4 disease: rapidly progressing neonatal intrahepatic cholestasis and early death

Background Clinical studies on progressive familial intrahepatic cholestasis (PFIC) type 5 caused by mutations in NR1H4 are limited. Methods New patients with biallelic NR1H4 variants from our center and all patients from literature were retrospectively analyzed. Results Three new patients were identified to be carrying five new variants. Liver phenotypes of our patients manifests as low-γ-glutamyl transferase cholestasis, liver failure and related complications. One patient underwent liver transplantation (LT) and survived, and two other patients died without LT. Nine other patients were collected through literature review. Twelve out of 13 patients showed neonatal jaundice, with the median age of onset being 7 days after birth. Reported clinical manifestations included cholestasis (13/13, 100%), elevated AFP (11/11, 100%), coagulopathy (11/11, 100%), hypoglycemia (9/13, 69%), failure to thrive (8/13, 62%), splenomegaly (7/13, 54%), hyperammonemia (7/13, 54%), and hepatomegaly (6/13, 46%). Six of 13 patients received LT at a median age of 6.2 months, and only one patient died of acute infection at one year after LT. Other 7 patients had no LT and died with a median age of 5 months (range 1.2-8). There were 8 patients with homozygous genotype and 5 patients with compound heterozygous genotype. In total, 13 different variants were detected, and 5 out of 12 single or multiple nucleotides variants were located in exon 5. Conclusions We identified three newly-diagnosed patients and five novel mutations. NR1H4-related PFIC typically cause progressive disease and early death. LT may be the only lifesaving therapy leading to cure. Supplementary Information The online version contains supplementary material available at 10.1186/s13023-024-03166-1.

transplant (LT) to save lives [4,[8][9][10].To assess phenotypic spectrum and clinical outcomes in NR1H4-related PFIC, we studied our patients in detail and reviewed previously reported patients in the literature.

Subjects
Our patients were all Chinese children referred to the Center for Pediatric Liver Disease, Children's Hospital of Fudan University from February 2016 to March 2023.Genetic testing, either whole-exome sequencing or liver panel sequencing, was performed in patients after excluding other causes of liver diseases (including infection, drug exposure, autoimmune hepatitis, and biliary atresia) [11].Cytomegalovirus infection was not excluded due to its high prevalence in Chinese infants.When other known inherited liver disorders were excluded, patients with two or biallelic NR1H4 pathogenic/likely pathogenic/uncertain significance (P/LP/US) variants were collected.P/LP/US variants were classified according to the American College of Medical Genetics (ACMG)/Association for Molecular Pathology (AMP) criteria [12].

Identification of biallelic NR1H4 variants in 3 new patients and in silico assessment
We identified 5 unique NR1H4 variants from 3 unrelated Chinese patients with low GGT intrahepatic cholestasis from our cohort.Among these, there were 3 missense variants, one nonsense variant, and one canonical splicing variant.All variants have not been previously reported in medical literatures (See Table 1).Three out of 5 variants were absent in gnomAD (c.505T > A/p.(Cys169Ser), c.1235T > C/p.(Leu412Pro), and c.1066 + 1G > A/p.?).The other two were present in gnomAD, with a population frequency of 0/1/251,458 (number of homozygotes/ allele count/allele number) for the c.688 C > T/p.(Arg-230Ter) variant and 0/1/31,410 (number of homozygotes/allele count/allele number) for the c.527G > A/p.(Arg176Gln) variant.MutationTaster predicted that the nonsense variant (c.688 C > T/p.(Arg230Ter)) may lead to nonsense-mediated mRNA decay (NMD).SpliceAI and varSEAK predicted that the canonical splicing variant (c.1066 + 1G > A/p.?) lead to loss of donor splice site, most likely leading to protein truncation.Three missense variants were predicted to be pathogenic by five pathogenicity prediction tools, and have no effect on pre-mRNA splicing.According to the ACMG/AMP criteria, all of the variants were classified as P/LP/US (Table 1).

Clinical features and outcome of 3 new patients with NR1H4-related cholestasis
All 3 male patients born at full-term with normal weight to non-consanguineous parents following uneventful pregnancies.The first child in family II died of unexplained liver disorders at the age of 7 months, whereas the other families did not have positive family history.All of them presented with jaundice during the first few days after birth.Normal growth and development were observed in patient 1(P1), and failure-to-thrive occurred in patients 2 (P2) and 3 (P3).Our patients have similar clinical features resembling previously reported patients, including low-GGT cholestasis, rapidly progressive liver failure/decompensated cirrhosis, vitamin K independent coagulopathy, and markedly elevated AFP levels (Table 2).Initial, pre-transplantation, or pre-death laboratory testing results were shown in Table 3.In addition, our patients had recurrent severe pneumonia, splenomegaly, and elevated urinary microalbumin.Two patients (P1 and P3) had hepatomegaly and hydrocele.
The patient P1 suffered massive ascites from decompensated cirrhosis prior to liver transplantation (LT).He had a positive plasma Epstein-Barr virus DNA from 2 months post-LT until the last visit.He also underwent a single surgery due to small bowel obstruction and rightsided diaphragmatic hernia one year after LT.The patient P3 presented with hyperammonemia due to acute liver failure (ALF) and his magnetic resonance of the brain showed minor abnormalities such as widened extracerebral space and cavum septum pellucidum.
Only one patient (P1) was treated by LT at the age of 4 months, and the other two patients died of infection at 3 months of age.Up to the latest assessment, P1 had normal liver function at one-year post-transplant.1).Eight patients had homozygous genotype and 5 patients had compound heterozygous genotype.In total, 13 different variants were detected, including 4 nonsense variants, 4 missense variants, 2 frameshift variants, one splice site variant, one in-frame insertion variant, and one large DNA fragment deletion variant.Overall, there were five variants (41.6%) in exon 5, two variants (16.7%) in exon 4, one variant (8.3%) in exon 6, one variant (8.3%) in exon 8, one variant (8.3%) in exon 9, one variant (8.3%) in intron 9 and one variant (8.3%) in exon 11 (Figure 1).

Liver pathological characteristics in NR1H4-related PFIC patients
Liver pathology was documented in 7 of 13 NR1H4related PFIC [8,9].Characteristic pathological features included cholestasis, steatosis, micro-nodular cirrhosis, hepatocellular ballooning, fibrous tissue proliferation, fibrosis, inflammatory cell infiltration, and proliferation of bile ducts.Immunohistochemical stainings of both bile salt export pump (BESP) and FXR proteins were absent, and the multi-drug resistance protein 3 (MDR3) expression was decreased or normal in all 7 patients.

Discussion
It was first discovered 7 years ago that the NR1H4 was responsible for PFIC [4].Previous studies showed that the NR1H4-associated PFIC had early-onset and rapid disease progression with high mortality [4,8].As only few patients have been reported, current understanding of PFIC5 caused by NR1H4 defect is limited.Therefore, we performed a retrospective analysis to obtain a better understanding of clinical phenotype and outcomes of PFIC5 caused by NR1H4 in our center and literature.
The FXR, encoded by NR1H4 gene, as the master regulator of BA homeostasis, regulates BA homeostasis, biliary BA secretion, and intestinal re-absorption [13][14][15].Compared with other PFIC patients, the PFIC5 patients caused by NR1H4 defect had significantly worse prognosis due to more rapid progression [8,16,17].All patients without LT died and survival with native liver has not been observed.Three new patients in our cohort exhibited similar clinical characteristics as published case [7,8].The liver phenotypes of all reported patients were extremely similar and presented as low GGT neonatal cholestasis with rapid progression to ALF (with/without related complications such as hypoglycemia, hyperammonemia, coagulopathy, hepatosplenomegaly, hydrothorax, and ascites) [8,9].While NR1H4 gene is predominantly expressed in liver and intestine, it also presents in kidney, spleen, heart, gallbladder, pancreas, adrenal glands, bone marrow, and other tissues [18][19][20][21].We further summarized the extrahepatic phenotypes of 13 patients.Of those, the failure to thrive was the most common finding.Other extrahepatic manifestations were also described, such as atrial septal defect, butterfly vertebra, decreased bone mineral density, intestinal obstruction, diaphragmatic hernia, inguinal hernia, and iris coloboma.Although NR1H4 is highly expressed in the intestine, recurrent or severe diarrhea has not been observed in patients with NR1H4-related disorder [8][9][10].
Notably, pharmacological therapy is typically not effective for NR1H4 disease [8].Those patients without LT died in early infancy, and the common causes of death included ALF, MODS, and severe infection [8].Therefore, LT may be the only curative option.Fortunately, 5 out of 6 patients are still alive after LT without serious postoperative complications, and with good clinical outcome during the follow-up.Only one patient died of acute infections one month after the transplant [9], this suggests that minimizing the risk of infection is the key to reduce morbidity and mortality associated with LT for PFIC5 patients [22,23].
All enrolled patients had poor prognosis with native liver.So, we were not able to analyze the relationship between genotypes and phenotype.However, we observed that 41.6% of all reported variants are located on the exon 5 of NR1H4 gene.Exon5 encodes a highly conserved DNA binding domain of FXR by binding to specific DNA sequences called hormone response elements, thereby possibly regulating other gene expression [24,25].More cases and further studies are needed to confirm whether exon 5 is a susceptible or hotspot region for NR1H4 gene mutation.

Conclusions
NR1H4-related PFIC is characterized by severe neonatal cholestasis, rapid progression to liver failure, and early death.LT might be the only lifesaving therapy that can lead to cure.At present, no severe complications of LT related to NR1H4 gene were observed, but longterm outcome of LT still needs to be validated in more patients.

Fig. 1
Fig. 1 Schematic presentation of the farnesoid X receptor (FXR) protein and locations of 12 unique single or multiple nucleotides variants identified in our cohort and literature.The exon 5 was depicted in red, and other exons of the NR1H4 gene are depicted in blue; FXR protein domains are shown in green

Table 2
Clinical features and outcomes of our 3 patients with NR1H4-related cholestasis

Table 3
Initial and pre-transplantation/pre-death laboratory testing of our 3 patients with NR1H4-related cholestasis